This project will provide a detailed understanding of the genetic mechanisms responsible for the spread of antibiotic resistance in Bacteroides. These anaerobic gram-negative bacteria are the predominant micro-organisms in the human gastro-intestianl tract, and function to maintain the normal microbial ecology of the bowel. In addition, Bacteroides are the most important isolates from anaerobic infections, and increasing antibiotic resistance in Bacteroides has complicated the treatment of these infections. Both plasmid and non-plasmid mediated transfer of antibiotic resistance has been described in Bacteroides, and indirect evidence for transposition of resistance markers has been reported. This project will utilize a novel gene transfer system recently developed to transfer plasmids from E. coli into Bacteroides. The system consists of a bifunctional shuttle vector capable of replication in both E. coli and Bacteroides, with transfer between E. coli and B. fragilis mediated by the broad host range plasmid RK2. This genetic system will be used to accomplish the following specific aims: 1) to locate and study the replication, transfer, and drug resistance genes on the Bacteroides R plasmid pCPl, 2) to characterize the mechanism of differential drug resistance gene expression in E. coli and Bacteroides, 3) to establish a gene library of Bacteroides DNA in E. coli, 4) to use the library to study the mechanism of the novel gene transfer systems that operate in Bacteroides in the absence of plasmids, and 5) to elucidate the mechanism of clindamycin resistance transposition in Bacteroides. This project will establish a genetic system in Bacteroides using current recombinant DNA technology. Such a system will facilitate the analysis not only of antibiotic resistance, but of physiology, metabolism, and virulence properties of these important anaerobes.